GTPases function as molecular switches to control a wide variety of cellular processes including many involved in inflammation and cancer. Most of the signaling events that are regulated by GTPases take place on the cytoplasmic leaflet of cellular membranes. GTPases, which lack intrinsic hydrophobic domains, are targeted to this location by a series of post-translational modifications of a C-terminal CAAX motif that includes prenylation, proteolysis, and carboxyl methylation. Some GTPases are further modified by palmitoylation. The most significant accomplishments of the work performed over the course of the first four years of GM55279 are the molecular cloning of one of the enzymes that modifies Ras, prenylcysteine carboxyl methyltransferase (pcCMT) and the determination of its subcellular localization exclusively in the ER and Golgi, the paradigm-shifting studies of Ras trafficking that the latter observation provoked, and elucidation of membrane targeting of Rho GTPases. The current application seeks to shift the focus from the biology of GTPases in inflammatory cells to studies of membrane targeting GTPase in general. The Specific Aims are: 1. Differential Tracking of H-Ras and K-Ras: using pulse-chase labeling and subcellular fractionation, microinjection followed by real-time imaging of fluorescent, initially unprocessed Ras proteins, dual color and dynamic (time-lapse, FRAP and FLIP) fluorescence analysis of GFP- tagged proteins, and S. cerevisiae mutants we will investigate the distinct pathways utilized by H- versus K-Ras; 2. Nuclear Targeting of Rac1: we will follow up on one of the unexpected and potentially significant observations of our Rho trafficking study, nuclear targeting of Rac1 masked by prenylation, by characterizing the cryptic nuclear localization signal and determining its functional relevance; 3. Ras Signaling on Intracellular Membranes: we will utilize GFP-Raf1-RBD, a novel reporter of Ras activation, to study Ras signaling events in situ in living cells and determine the kinetics, pathways and significance of signaling on internal membranes using a variety of systems, including C. elegans to assess in vivo the functional capacity of endomembrane-restricted Ras. The information gained from the proposed studies will not only help elucidate an important area of GTPase biology but they may give insights into drug development for the myriad of pathologic conditions, such as oncogenesis and inflammation, that are controlled, in part, by GTPases.